Vaccines for the protection of animals against theileria infection

ABSTRACT

This invention relates to the development of a vaccine against Theileria parva, which is a protozoan parasite infecting cattle in Africa. The invention specifically relates to the use of the 67 kDa glycoprotein from the surface of the T. parva sporozoite as an immunogen for inducing immunoprotection against T. parva in bovine species. This 67 kDa antigen is produced using recombinant genetics. Plasmids containing nucleic acid segments encoding the antigen, host cells containing the nucleic acid segments and recombinant methods for producing the antigen are part of this invention.

This is a continuation of application Ser. No. 07/365,999 filed Jun. 14,1989, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the development of a vaccine against Theileriaparva, which is a protozoan parasite infecting cattle in Africa. Theinvention specifically relates to the use of the 67 kDa glycoproteinfrom the surface of the T. parva sporozoite as an immunogen for inducingimmunoprotection against T. parva in bovine species. This 67 kDa antigenis produced using recombinant genetics. Plasmids containing nucleic acidsegments encoding the antigen, host cells containing the nucleic acidsegments and recombinant methods for producing the antigen are part ofthis invention.

This invention also relates to the development of live vaccines againstTheileria parva, which is a protozoan parasite infecting cattle inAfrica. The invention specifically relates to the use of live attenuatedstrains of Salmonella typhimurium and vaccinia virus carrying the geneencoding the 67 kDa glycoprotein from the surface of the T. parvasporozoite as an immunogen for inducing immunoprotection against T.parva in bovine species. Construction of attenuated S. typhimurium,vaccinia viruses and plasmids containing nucleic acid segments encodingthe antigen are a part of this invention. Finally this inventionprovides methods for immunoprotecting animals against T. parvainfection.

The protozoan parasite, Theileria parva, which is transmitted by theixodid tick, Rhipicephalus appendiculatus, causes East Coast fever(ECF), a disease of cattle which continues to exert severe limitationson the development of the livestock industry in much of Eastern andCentral Africa. T. parva is divided into three sub-types, T. parvaparva, T. parva bovis and T. parva lawrencei. This classification isbased on certain epidemiological and behavioral characteristics of theparasites. T. parva lawrencei originates from buffalo and is highlypathogenic to cattle. T. parva bovis and T. parva parva are maintainedbetween cattle and differ in pathogenicity. Despite this classificationthere is no definitive proof for sub-speciation of T. parva. For thepurposes of this document, T. parva therefore encompasses all threesub-types which are themselves heterogenous in nature.

Infection with T. parva is initiated when sporozoites are inoculatedinto the mammalian hosts by the tick during feeding. The sporozoitesenter lymphocytes where they mature into schizonts. The intralymphocyticschizonts later develop to merozoites which enter erythrocytes to becomepiroplasms which are ingested by ticks feeding on the parasitized host.Gametes which develop from the piroplasms fuse to initiate the parasitelife cycle in the tick which culminates in the production of sporozoitesin the tick salivary gland.

Information Disclosure

Theileria infections of cattle have been recognized as a cause of aserious and often lethal disease of cattle since the early 1900's. Areview of the disease can be found in Irvin A. D. and Morrison W. I.Immune Responses in Parasitic Infection: Immunology, immunopathology,and immunoprophylaxis Vol. III, Ed. E. J. L. Soulsby CRC Press Inc.Florida, 1987, pp 223-274. T. parva infection results in a high rate ofmortality; however, some animals recover from the infection and aresubsequently immune to homologous challenge. At present, it is possibleto induce immunity by infecting cattle with sporozoites andsimultaneously administering oxytetracycline. Radley, D. E., et al.,1975, East Coast fever: 1. Chemoprophylactic immunization of cattleagainst Theileria parva (Muguga) and five theilerial strains. VetParasitol. 1:35-41; and Radley et al., 1975, East Coast fever: 3.Chemoprophylactic immunization of cattle using oxytetracycline and acombination of theilerial strains Vet. Parasitol. 1, 51-60. However,this method of immunization affords protection against only a limitednumber of different T. parva isolates, Radley, D. E. et al., 1975. EastCoast fever. 2. Cross-immunity trials with a Kenya strain of Theilerialawrencei. Vet. Parasitol. 1: 43-50; Cunningham, M. P., et al., 1974,Theileriosis: The exposure of immunized cattle in a Theileria lawrenceienzootic area. Trop. Anim. Hlth. 6, 39-43; and Irvin, A. D., et al.,1983, Immunization against East Coast fever: correlation betweenmonoclonal antibody profiles of Theileria parva isolates and crossimmunity in vivo. Res. Vet. Sci. 35:341-346].

It has been shown that animals which have developed immunity to T. parvaexhibit antibody responses against proteins of the sporozoite stage(Musoke, A. J., et al., 1982, Bovine immune response to Theileria parva:neutralizing antibodies to sporozoites, Immunology 45:663-668) as wellas a cell-mediated response against the schizont stage (Eugui, E. M. andEmery, D. L., 1981, Genetically restricted cell-mediated cytotoxicity incattle immune to Theileria parva. Nature 290, 251-254). There isevidence that antibodies raised against sporozoite antigens can blockinfectivity in vitro in a non-isolate specific manner (Musoke, A. J., etal., 1984, Evidence for a common protective antigenic determinant onsporozoites of several Theileria parva strains. Immunology 52, 231-238;and Dobbelaere, D. A. E., 1984, Monoclonal antibody neutralizes thesporozoite stage of different Theileria parva isolates, ParasiteImmunol. 6:361-370). However, the extent of protection conferred incattle by this humoral response, and the role played by particularsporozoite antigens, have not yet been reliably evaluated.

The 67 kDa antigen that is the subject of this invention was previouslyknown to be an antigen on the sporozoite surface (Dobbelaere, D. A. E.et al., 1985, Identification of a surface antigen on Theileria parvasporozoites by monoclonal antibody, Proc. Natl. Acad. Sci., U.S.A.82:1771-1775; and Dobbelaere, D. A. E. et al., 1985, Theileria parva:Expression of a sporozoite surface coat antigen, ExperimentalParasitology, 60:90-25).

DESCRIPTION OF FIGURES

FIG. 1. DNA and inferred amino acid sequence of the sporozoite 67 kDagene.

The DNA sequence corresponding to the open reading frame up to the polyAtail is shown. The position of important restriction enzyme sites ismarked. The site of cleavage of the signal sequence to release themature sporozoite antigen was predicted according to Perlman andHalvorson, 1983, J.Mol.Biol. 167:391-409. As judged by these "rules" thecleavage site could be at any one of three positions; between residues16-17, 17-18 and 18-19.

FIG. 2. The recombinant vector phTpp(mug)-p67 sp comprising thesporozoite 67 kDa antigen that has been deposited.

FIG. 3. Construction of an expression plasmid for production of the 67kDa antigen in E. coli involving the use of a full length cDNA.

FIG. 4. Assembly of a prokaryotic expression plasmid from Theileria cDNAand genomic DNA.

FIG. 5. Construction of an expression plasmid for production of the 67kDa antigen in mouse cells using full length cDNA.

FIG. 6. Construction of an expression plasmid for production of the 67kDa antigen in mouse cells using sequences assembled from cDNA andgenomic DNA.

SUMMARY OF THE INVENTION

This invention provides for a composition of substantially pureTheileria parva sporozoite surface glycoprotein of about 67 kDa ormodifications thereof having immunological crossreactivity withTheileria sera said glycoprotein having the amino acid sequence setforth in FIG. 1. When this glycoprotein is produced by bacteria whichhave been genetically altered to express the glycoprotein thecomposition will be devoid of carbohydrate side chains ordinarilyattached by eukaryote cells. It is preferred that the compositions ofthis invention are substantially free of other proteins or polypeptidesof Theileria origin. By Theileria origin, we refer to proteins derivedfrom or originating from species of this genus of protozoa.

This invention also provides for recombinant DNA sequences comprising aDNA segment encoding a Theileria parva sporozoite surface glycoproteinof about 67 kDa or modifications thereof having immunologicalcrossreactivity with Theileria sera, said glycoprotein having the aminoacid sequence set forth in FIG. 1. It is also disclosed herein, that theabove segment may be recombined in positions adjacent to either DNAsequences derived from vaccinia virus or adjacent to DNA sequencesderived from Salmonella type bacteria. The preferred salmonella typebacteria are Salmonella typhimurium.

The DNA segment described above may also be made a part of a recombinantDNA plasmid. Such plasmids would preferably direct the expression of theglycoprotein in a bacterial or eukaryote host cell. The preferred hostcells are selected from the group consisting of Escherichia coli andSalmonella typhimurium.

This invention also provides for vaccines for inducing immunoprotectionin animals against infections with species of Theileria comprising atleast one active ingredient selected from the group consisting of asubstantial pure sporozoite surface glycoprotein of about 67 kDa; amodification of a said glycoprotein having immunological crossreactivitywith Theileria sera; a sequence of DNA encoding said glycoprotein; and asequence of DNA encoding said modification of said glycoprotein; whereinthe glycoprotein has the amino acid sequence set forth in FIG. 1. Thesevaccines may also include compositions comprising live Salmonellabacteria capable of expressing the Theileria parva sporozoite surfaceglycoprotein as described above. These salmonella bacteria may carry theglycoprotein gene either as a stably maintained expression plasmid or asa segment of DNA integrated into its chromosome.

Alternatively, the vaccine may comprise vaccinia virus modified toexpress in infected cells the Theileria parva sporozoite surfaceglycoprotein of about 67 kDa or a modification thereof as defined above.

The vaccines of this invention are preferably protective againstinfection from Theileria parva.

There is also disclosed herein a method for protecting animals frominfections of species of Theileria comprising the administration of aneffective amount of a vaccine comprising at least one active ingredientselected from the group consisting of a substantially pure sporozoitesurface glycoprotein of about 67 kd; a modification of a saidglycoprotein having immunological crossreactivity with Theileria sera; asequence of DNA encoding said glycoprotein; or a sequence of DNAencoding said modification of said glycoprotein; wherein theglycoprotein has the amino acid sequence set forth in FIG. 1. Saidmethod can be conducted with any of the vaccines described above orcombinations thereof.

A culture deposit of E.coli containing recombinant plasmids encoding theTheileria 67 kDa antigen has been made. The culture was deposited withthe National Collections of Industrial Bacteria Limited (NCIMB) at 15Abbey Road, Aberdeen AB9 8DG, Scotland, U.K., on May 15, 1989 and givenan Accession Number of NCIMB 40147. See FIG. 2 for a restriction enzymemap of the deposited plasmid, phTpp (mug)-p67sp.

DETAILED DESCRIPTION

This invention provides for means of producing the 67 kDa antigen inquantities that will permit large scale vaccination of cattle against T.parva. The current immunization procedure of administering the infectiveparasite followed by drug treatment is not practical. The procedurerequires a good veterinary infrastructure which is not generallyavailable. An expensive liquid nitrogen facility is required for storageof the parasite. Furthermore, the parasites may also become anaccidental source of infection to the animals.

A purified 67 kDa antigen is more practical and effective as an activecomponent in a vaccine. At least one antigenic determinant on the 67 kDaantigen of T. parva parva (Muguga) is conserved since one monoclonalantibody will in an in vitro assay neutralise sporozoites from differentisolates of the parasites. The 67 kDa antigen should therefore affordprotection against all sub-types of T. parva.

The large scale isolation of sporozoites as a source of the 67 kDaantigen is not a practical means of producing a vaccine as thesporozoites must be isolated from the dissected salivary glands ofticks. In addition tick infection rates vary considerably making itdifficult to consistently obtain large numbers of sporozoites.

Rather than extract the 67 kDa antigen directly from sporozoites, onecan use recombinant genetics to facilitate the production of theTheileria antigen. One standard method would involve the introduction ofDNA encoding the 67 kDa sporozoite surface antigen into a suitable hostcell, followed by induction of that cell to produce large amounts of theselected protein. This invention embraces such molecular geneticmanipulations. The following descriptions will detail the variousmethods available to express genes encoding Theileria antigens, and isfollowed by specific examples of preferred methods.

An alternative method involves the administration of live S. typhimuriumor vaccinia virus that contains the gene encoding the 67 kDa antigen.Live vaccines will induce a potent immune response against T. parva,without the need for purification of the 67 kDa antigen. The followingdescriptions also detail various methods available to express genesencoding Theileria antigens in Salmonella typhimurium and vaccinia, andis followed by specific examples of preferred methods.

A. General Methods

Much of the nomenclature and general laboratory procedures required inthis application can be found in Maniatis, T. et al., MolecularCloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1982. The manual is hereinafter referred to as "Maniatis".

All E. coli and S. typhimurium strains are grown in Luria broth (LB) orM9 medium supplemented with glucose and acid-hydrolyzed casein aminoacids. Strains with resistance to antibiotics are maintained at the drugconcentrations described in Maniatis.

Vaccinia virus is grown in suitable cultured mammalian cells such as theHeLA S3 spinner cells, as described by Mackett, Smith and Moss, "Theconstruction and characterization of Vaccinia Virus RecombinantsExpressing Foreign Genes" in "DNA cloning Vol. II. A practicalapproach", Ed. D. M. Glover, IRL Press, Oxford, pp 191-211.

All enzymes are used according to the manufacturer's instructions.

cDNA libraries were constructed in bacteriophage lambda gt11. Phage arepackaged in vitro, and recombinant phage were analyzed by plaquehybridization as described in Benton and Davis, Science 196: 180-1821977 and in Maniatis. Lambda gt11 DNA and the in vitro packaging systemcan be obtained from Promega Biotec.

DNA was sequenced using the dideoxy procedure of Sanger, F. et al.,1977, PNAS 74: 5463-5467 or by the modified T7 DNA polymerase procedureof Tabor and Richardson, 1987, PNAS, 84: 4767-4771. The latter wasobtained as a kit (Sequanase) from U.S. Biochemicals and used accordingto the manufacturer's instruction.

Polynucleotide sizes are given in either kilo-base pairs (kb) orbasepairs (bp). These are either estimates derived from agarose gelelectrophoresis or actual sizes determined by DNA sequencing. B.Theileria 67 kDa Sporozoite Surface Antigen cDNA

The first step in obtaining expression of the gene encoding the 67 kDaantigen is to obtain the DNA sequence coding for the protein from cDNAclones. A full length cDNA is then cloned into an expression vectorwhich is capable of directing efficient transcription and translation ofthe gene.

The method for obtaining cDNA has been described generally in Maniatis.Parasite messenger RNA is converted into cDNA as described by Gubler andHoffman, 1983, Gene, 25: 263-269. A library of clones is prepared byadding EcoRI linkers to the cDNA, ligating the cDNA to EcoRI digestedDNA arms of lambda gt11 and packaging the ligated DNA into viable phageparticles. The clones containing antigen cDNA are detected byhybridization with probes of labelled synthetic oligonucleotidescomplementary to portions of the cDNA sequence (Wallace, R. B., et al.,1981, Nucleic Acids Res. 9: 879-894), followed by restriction enzymeanalysis and DNA sequencing.

The synthetic oligonucleotide probes are single-stranded DNA molecules,typically between 10 and 50 nucleotides in length. FIG. 1 provides thenucleic acid sequence of the 67 kDa antigen from which suitableoligonucleotide probes may be derived.

Oligonucleotides for use as probes are chemically synthesized accordingto the solid phase phosphoramidite triester method first described byBeaucage, S. L. and Caruthers, M. H., 1981, Tetrahedron Letts., 22(20):1859-1862 using an automated synthesizer, as described inNeedham-VanDevanter, D. R., et al., 1984, Nucleic Acids Res.,12:6159-6168. Purification of oligonucleotides is by either nativeacrylamide gel electrophoresis or by anion-exchange HPLC as described inPearson, J. D. and Regnier, F. E., 1983, J. Chrom., 255:137-149.

The sequence of the synthetic oligonucleotides can be verified using thechemical degradation method of Maxam, A. M. and Gilbert, 1980, in W.,Grossman, L. and Moldave, D., eds. Academic Press, New York, Methods inEnzymology, 65:499-560.

C. Expression in Prokaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding Theileria antigens in a prokaryotic system, it is essential toconstruct expression plasmids which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.Examples of regulatory regions suitable for this purpose in E. coli arethe promoter and operator region of the E. coli tryptophan biosyntheticpathway as described by Yanofsky, C., 1984, J. Bacteriol., 158:1018-1024and the leftward promoter of phage lambda (P_(L)) as described byHerskowitz, I. and Hagen, D., 1980, Ann. Rev. Genet., 14:399-445.

Expression systems for expressing the 67 kDa antigens are availableusing E. coli, Bacillus sp. (Palva, I et al., 1983, Gene 22:229-235;Mosbach, K. et al., Nature, 302:543-545 and Salmonella. E. coli andSalmonella systems are preferred.

The 67 kDa antigen produced by prokaryote cells will not be glycosylatedand the antigen may not necessarily fold properly. During purificationfrom E. coli, the expressed antigen protein may first be denatured andthen renatured. This can be accomplished by solubilizing the bacteriallyproduced proteins in a chaotropic agent such as guanidine HCl andreducing all the cysteine residues with a reducing agent such as beta-mercaptoethanol. The protein is then renatured, either by slow dialysisor by gel filtration. U.S. Pat. No. 4,511,503.

Detection of the expressed antigen is achieved by methods known in theart as radioimmunoassays, or Western blotting techniques orimmunoprecipitation. Purification from E. coli can be achieved followingprocedures described in U.S. Pat. No. 4,511,503.

D. Expression in S. typhimurium

A serious limitation to the expression of heterologous gene productsfrom plasmids in Salmonella is that the commonly used plasmid cloningvectors are inherently unstable. To circumvent this, the foreign genecan be incorporated into a nonessential region of the host chromosome.This is achieved by first inserting the gene into a plasmid such that itis flanked by regions of DNA homologous to the insertion site in theSalmonella chromosome. After introduction of the plasmid into the S.typhimurium, the foreign gene is incorporated into the chromosome byhomologous recombination between the flanking sequences and chromosomalDNA.

An example of how this could be achieved is based on the his operon ofSalmonella. Two steps are involved in this process. Firstly, a segmentof the his operon must be deleted in the Salmonella strain selected asthe carrier. Secondly, a plasmid carrying the deleted his regiondownstream of the gene encoding the 67 kDa antigen is transformed intothe his Salmonella strain. Integration of both the his sequences and thegene encoding the 67 kDa antigen occurs, resulting in recombinantstrains which can be selected as His³⁰.

Detection of the expressed antigen is achieved by methods known in theart such as radioimmunoassays, Western blotting or immunoprecipitation.

The Salmonella strain used in the vaccine is derived from strainsnormally virulent for cattle. Specific attenuation of the strains renderthe bacteria avirulent but still capable of inducing a potent immuneresponse after inoculation into cattle. An example of such a strain isthe aro A mutant of S. typhimurium (Smith B. P. et al., Am. J. Vet. Res.45:59-66).

E. Synthesis of the Theileria Antigen Proteins in Eukaryotes

The Theileria 67 kDa antigen is a glycoprotein. Prokaryotic expressionsystems generally lack the ability to glycosylate eukaryotic proteins.Therefore, it is often advantageous to express a particular protein inan eukaryotic system, especially when a significant proportion of theimmunogenicity resides in the carbohydrate portion of the antigen.

1. Expression in recombinant vaccinia virus-infected cells

The gene encoding the 67 kDa antigen is inserted into a plasmid designedfor producing recombinant vaccinia, such as pG562, Langford, C. L., etal., 1986, Mol. Cell. Biol. 6:3191-3199. This plasmid consists of acloning site for insertion of foreign genes, the P7.5 promoter ofvaccinia to direct synthesis of the inserted gene, and the vaccinia TKgene flanking both ends of the foreign gene.

When the plasmid containing the 67 kDa antigen gene is constructed, thegene can be transferred to vaccinia virus by homologous recombination inthe infected cell. To achieve this, suitable recipient cells aretransfected with the recombinant plasmid by standard calcium phosphateprecipitation techniques into cells already infected with the desirablestrain of vaccinia virus, such as Wyeth, Lister, WR or Copenhagen.Homologous recombination occurs between the TK gene in the virus and theflanking TK gene sequences in the plasmid. This results in a recombinantvirus with the foreign gene inserted into the viral TK gene, thusrendering the TK gene inactive. Cells containing recombinant viruses areselected by adding medium containing 5-bromodeoxyuridine, which islethal for cells expressing a TK gene.

Confirmation of production of recombinant virus can be achieved by DNAhybridization using cDNA encoding the 67 kDa antigen and byimmunodetection techniques using antibodies specific for the expressedprotein. Virus stocks may be prepared by infection of cells such as HeLAS3 spinner cells and harvesting of virus progeny.

2. Expression in Yeast

Synthesis of heterologous proteins in yeast is well known and described.Methods in Yeast Genetics, Sherman, F., et al., Cold Spring HarborLaboratory, (1982) is a well recognized work describing the variousmethods available to produce the Theileria 67 kDa antigen in yeast.

For high level expression of a gene in yeast, it is essential to connectthe gene to a strong promoter system as in the prokaryote and also toprovide efficient transcription termination/polyadenylation sequencesfrom a yeast gene. Examples of useful promoters include GALl,lO(Johnson, M., and Davies, R. W., 1984, Mol. and Cell. Biol.,4:1440-1448) ADH2 (Russell, D., et al., 1983, J. Biol. Chem.,258:2674-2682), PHO5 (EMBO J. 6:675-680, 1982), and MFαl. A multicopyplasmid with a selective marker such as Leu-2, URA-3, Trp-l, and His-3is also desirable.

The MFαl promoter is preferred. The MFαl promoter, in a host of the αmating-type is constitutive, but is switched off in diploids or cellswith the a mating-type. It can, however, be regulated by raising orlowering the temperature in hosts which have a ts mutation at one of theSIR loci. The effect of such a mutation at 35° C. on an α type cell isto turn on the normally silent gene coding for the α mating-type. Theexpression of the silent a mating-type gene, in turn, turns off the MFαlpromoter. Lowering the temperature of growth to 27° C. reverses thewhole process, i.e., turns the a mating-type off and turns the MFαl on(Herskowitz, I. and Oshima, Y., 1982, in The Molecular Biology of theYeast Saccharomyces, (eds. Strathern, J. N. Jones, E. W., and Broach, J.R., Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., pp.181-209.

The polyadenylation sequences are provided by the 3'-end sequences ofany of the highly expressed genes, like ADHl, MFαl, or TPI (Alber, T.and Kawasaki, G., 1982, J. of Mol. & Appl. Genet. 1:419-434.

A number of yeast expression plasmids like YEp6, YEpl3, YEp4 can be usedas vectors. A gene of interest can be fused to any of the promoters invarious yeast vectors. The above-mentioned plasmids have been fullydescribed in the literature (Botstein, et al., 1979, Gene, 8:17-24;Broach, et al., 1979, Gene, 8:121-133).

Two procedures are used in transforming yeast cells. In one case, yeastcells are first converted into protoplasts using zymolyase, lyticase orglusulase, followed by addition of DNA and polyethylene glycol (PEG).The PEG-treated protoplasts are then regenerated in a 3% agar mediumunder selective conditions. Details of this procedure are given in thepapers by J. D. Beggs, 1978, Nature (London), 275:104-109; and Hinnen,A., et al., 1978, Proc. Natl. Acad. Sci. U.S.A., 75:1929-1933. Thesecond procedure does not involve removal of the cell wall. Instead thecells are treated with lithium chloride or acetate and PEG and put onselective plates (Ito, H., et al., 1983, J. Bact., 153:163-168).

The Theileria 67 kDa sporozoite surface antigen can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques orradioimmunoassays.

3 Expression in Cell Cultures

The Theileria 67 kDa antigen cDNA can be ligated to various expressionvectors for use in transforming host cell cultures. The vectorstypically contain gene sequences to initiate transcription andtranslation of the Theileria antigen gene. These sequences need to becompatible with the selected host cell. In addition, the vectorspreferably contain a marker to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase ormetallothionein. Additionally, a vector might contain a replicativeorigin.

Illustrative of cell cultures useful for the production of the Theileriaantigen are cells of insect or mammalian origin. Mammalian cell systemsoften will be in the form of monolayers of cells although mammalian cellsuspensions may also be used. Illustrative examples of mammalian celllines include VERO and HeLa cells, Chinese hamster ovary (CHO) celllines, WI38, BHK, COS-7 or MDCK cell lines.

As indicated above, the vector, e.g., a plasmid, which is used totransform the host cell, preferably contains DNA sequences to initiatetranscription and sequences to control the translation of the antigengene sequence. These sequences are referred to as expression controlsequences. When the host cell is of insect or mammalian originillustrative expression control sequences are obtained from the SV-4Opromoter (Science, 222:524-527, 1983), the CMV I. E. Promoter (Proc.Natl. Acad. Sci. 81:659-663, 1984) or the metallothionein promoter(Nature 296:39-42, 1982). The cloning vector containing the expressioncontrol sequences is cleaved using restriction enzymes and adjusted insize as necessary or desirable and ligated with cDNA coding for theTheileria 67 kDa antigen by means well known in the art.

As with yeast, when higher animal host cells are employed,polyadenlyation or transcription terminator sequences from knownmammalian genes need to be incorporated into the vector. An example of aterminator sequence is the polyadenlyation sequence from the bovinegrowth hormone gene. Sequences for accurate splicing of the transcriptmay also be included. An example of a splicing sequence is the VP1intron from SV40 (Sprague, J. et al., 1983, J. Virol. 45: 773-781).

Additionally gene sequences to control replication in the host cell maybe incorporated into the vector such as those found in bovine papillomavirus type-vectors. Saveria-Campo, M., 1985, "Bovine Papilloma virus DNAa Eukaryotic Cloning Vector" in DNA Cloning Vol.II a Practical ApproachEd. D. M. Glover, IRL Press, Arlington, Va. pp. 213-238.

The host cells are competent or rendered competent for transformation byvarious means. There are several well-known methods of introducing DNAinto animal cells. These include: calcium phosphate precipitation,fusion of the recipient cells with bacterial protoplasts containing theDNA, treatment of the recipient cells with liposomes containing the DNA,DEAE dextran, electropora-tion and micro-injection of the DNA directlyinto the cells.

The transformed cells are cultured by means well known in the art.Biochemical Methods in Cell Culture and Virology, Kuchler, R. J.,Dowden, Hutchinson and Ross, Inc., (1977). The expressed theilerialantigen is isolated from cells grown as suspensions or as monolayers.The latter are recovered by well known mechanical, chemical or enzymaticmeans.

Isolation of the Theileria antigen can be accomplished by lysing thehost cells with detergents. Further purification is accomplished byaffinity, ion-exchange or gel filtration chromatography using theprocedures generally used to purify the antigen from sporozoites. (Seegenerally, Pharmacia Fine Chemicals literature: Affinity ChromatographyPrinciples and Methods, Ion Exchange Chromatography Principle andMethods and Gel Filtration Theory and Practice.)

F. Vaccines Against Theileria parva

a) General, non-vectored

A vaccine prepared utilizing the Theileria 67 kDa antigen or immunogenicequivalents thereof can comprise: (a) a crude cell extract of T.parvasporozoites or a suspension of chemically fixed sporozoites; (b) a crudeextract of cells recombinantly altered to express the Theileria 67 kDaantigen or a chemically-fixed suspension of such cells; (c) a partiallyor completely purified Theileria antigen preparation. The antigenproduced by recombinant DNA technology is preferred because it is moreeconomical than the other sources and is more readily purified in largequantities. The 67 kDa antigen can be prepared in unit dose form bywell-known procedures. The vaccine can be administered intramuscularlyor subcutaneously. For parenteral administration, such as bysubcutaneous injection, the antigen may be combined with a suitablecarrier. For example, it may be administered in water, saline orbuffered vehicles with or without various adjuvants or immunomodulatingagents such as aluminum hydroxide, aluminum phosphate, aluminumpotassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon,water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide,bacterial endotoxin, lipid, Propionobacterium acnes, (Corynebacteriumparvum), Bordetella pertussis, polyribonucleotides, sodium alginate,lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole,DEAE-dextran, Iscoms (Morein et al., (1984), Nature 408: 457-460),blocked copolymers or other synthetic adjuvants. Such adjuvants areavailable commercially from various sources, for example, Merck Adjuvant6 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants areFreund's Incomplete Adjuvant (Difco Laboratories, Detroit, Mich.) andMPL+TDM Emulsion (RIBBI Immunochem Research Inc. U.S.A.). Otherimmuno-stimulants include interleukin 1, interleukin 2 andinterferon-gamma. These proteins can be provided with the vaccine ortheir corresponding genetic sequence provided as a functional operonwith a recombinant vaccine system such as vaccinia virus. The proportionof antigen and adjuvant can be varied over a broad range so long as bothare present in effective amounts. For example, aluminum hydroxide can bepresent in an amount of about 0.5% of the vaccine mixture (Al₂ O₃basis). On a per-dose basis, the concentration of the antigen can rangefrom about 1.0 ug to about 100 mg per bovine host. A preferable range isfrom about 100 ug to about 3.0 mg per unit dose. A suitable dose size isabout 1-10 ml, preferably about 1.0 ml. Accordingly, a dose forsubcutaneous injection, for example, would comprise 1 ml containing 1.0mg of antigen and 3 mg of saponin.

For the initial vaccination of immunologically naive cows, a regimen ofbetween 1 and 4 unit doses can be used with the injections spaced outover a 2- to 6-week period. Typically, a two-dose regimen is used. Thesecond dose of the vaccine then should be administered some weeks afterthe first dose, for example, about 4 to 8 weeks later. Animals that havebeen previously exposed to Theileria parva or have received colostralantibodies from the mother may require booster injections. The boosterinjection is preferably timed to coincide with times of maximalchallenge. Different immunization regimes may be adopted depending onthe prevailing climate of the region. Semi-annual revaccination isrecommended for breeding animals. Steers and bulls may be revaccinatedat any time. Also, cows can be revaccinated before breeding. Calves maybe vaccinated at about 2 to 3 months after birth, again at 4 to 6months, and yearly or preferably semi-annually thereafter.

The vaccine may also be combined with vaccines for other diseases toproduce multivalent vaccines. It may also be combined with othermedicaments, for example, antibiotics. A pharmaceutically effectiveamount of the vaccine can be employed with a pharmaceutically acceptablecarrier or diluent understood to be useful for the vaccination ofanimals such as swine, cattle, sheep, goats, and other mammals. Theseadditives including adjuvants are referred to as "injectables ofnon-Theileria parva origins." Other vaccines may be prepared accordingto methods well-known to those skilled in the art as set forth, forexample, in I. Tizard, An Introduction to Veterinary Immunology, 2nd Ed,1982, which is incorporated herein by reference.

b) S. typhimurium

A vaccine prepared utilizing the gene encoding the 67 kDa antigenexpressed in S. typhimurium can comprise either a) live attenuated S.typhimurium harboring a stable plasmid containing the gene encoding the67 kDa antigen in a form suitable for expression of the gene or b) liveattenuated S. typhimurium in which the gene encoding the 67 kDa antigenhas been incorporated into the host chromosome in a form suitable forexpression of the gene.

For the initial vaccination of immunologically naive cows, a typicalregimen would consist of two doses of 10⁹ bacteria/dose delivered 1 weekapart. The intramuscular route is preferred as this would minimizerelease of the bacterium into the environment.

c) Vaccinia virus

A vaccine prepared utilizing the gene encoding the 67 kDa antigenincorporated into vaccinia virus would comprise stocks of recombinantvirus where the gene encoding the 67 kDa antigen is integrated into thegenome of the virus in a form suitable for expression of the gene.

For the initial vaccination of immunologically naive cows, a typicalregimen would consist of two doses of 4×10⁸ plaque forming units (p.f.u) of virus, inoculated intra-muscularly four weeks apart.

G. Definitions

The phrase "cell culture" refers to the containment of growing cellsderived from a multi-cellular plant or animal which allows for the cellsto remain viable outside the original plant or animal.

The term "microorganism" includes both single cellular prokaryote andeukaryote organisms such as bacteria, actinomycetes and yeast.

The term "plasmid" refers to an autonomous self-replicating circular DNAmolecule and includes both the expression and nonexpression types. Wherea recombinant microorganism or cell culture is described as hosting an"expression plasmid", this includes both extrachromosomal circular DNAmolecules and DNA that has been incorporated into the hostchromosome(s). Where a plasmid is being maintained by a host cell, theplasmid is either being stably replicated by the cells during mitosis asan autonomous structure or is incorporated within the host's genome.

The phrase "substantially pure," in the context of the Theileria 67 kDaantigen, refers to compositions containing the Theileria 67 kDasporozoite surface antigen or protein derivative. Substantially pureantigen may be contaminated with low levels of protein from theTheileria parva sporozoites, or from recombinant host cell constituents.The amount of contaminating proteins is such that the vaccinated animalwill not respond with significant levels of antibodies against saidcontaminants. Typically, the antigen preparation will be pure to atleast 75%, preferably at a purity in excess of 95%, and most preferablyin excess of 98%.

The phrase "Theileria 67 kDa sporozoite surface antigen" unlessotherwise stated, is meant to include both the naturally occurringsporozoite surface glycoprotein, and protein derivatives embracingdeletions and changes in the amino acid sequence and carbohydrate sidechains such that they appear to the immune system as functionalequivalents for purposes of protection from Theileria infection. Thesenon-natural derivatives are also known as "immunogenic equivalents".Those of skill will readily recognize that it is only necessary toexpose a mammal to appropriate epitopes in order to elicit effectiveimmunoprotection. The epitopes are typically segments of amino acidswhich are a small portion of the whole protein. Using recombinantgenetics it is simple and routine to alter a natural protein's primarystructure to create derivatives embracing epitopes that are identical toor substantially the same as (immunologically equivalent to) thenaturally occurring epitopes. Such proteins would exhibit crossreactivity with the antisera produced against the natural 67 kDaantigen. These protein derivatives may include peptide fragments, aminoacid substitutions, amino acid deletions and amino acid additions withinthe natural amino acid sequence for the Theileria 67 kDa antigens. Forexample, it is known in the protein art that certain amino acid residuescan be substituted with amino acids of similar size and polarity withoutan undue effect upon the biological activity of the protein or itsantigenicity. In the primary sequence of the theilerial antigen, thefollowing residues are generally considered to be interchangeable innon-critical regions: (1) alanine, leucine, isoleucine, valine andproline are interchangeable, (2) phenylalanine and tryptophan areinterchangeable, (3) serine, threonine and tyrosine are interchangeable,(4) asparagine and glutamine are interchangeable, (5) lysine, arginine,histidine and ornithine are interchangeable, and (6) aspartic acid andglutamic acid are interchangeable.

The phrase "DNA sequence" refers to a single or double stranded DNAmolecule composed of nucleotide bases, adenosine, thymidine, cytosineand guanosine.

The phrase "suitable host" refers to a cell culture or microorganismthat is compatible with a recombinant plasmid and will permit theplasmid to replicate, to be incorporated into its genome or to beexpressed.

The phrase "Theileria sera" refers to blood serum containing antibodiesreactive with native 67 kDa antigen.

EXAMPLE 1 Cloning of the 67 kDa glycoprotein gene from Theileriasporozoite mRNA A. Parasite Stabilates

T. parva sporozoites are derived from stabilate Muguga 10. For piroplasmpreparation, calves, 6 to 12 months of age, are infected by inoculationwith a sporozoite stabilate prepared as previously described inCunningham, M. P., et al., 1973, Int. J. Parasit. 3:583-587.

B. Construction of a cDNA library in lambda gt 11

The following procedure details the isolation of Theileria cDNA encodingthe 67 kilodalton glycoprotein of the sporozoites using a synthetic DNAprobe complementary to the 5' end of the gene which codes for theN-terminal amino acid sequence of the protein. The DNA sequence andinferred amino acid sequence of the Theileria antigen gene are providedin FIG. 1.

Dissected salivary glands from T. parva parva (Muguga) infected ticksfed for three days on rabbits are flash frozen in liquid nitrogen. TotalRNA is isolated from the salivary glands using the hot phenol/SDS methodas described by Cordingley, J. S. et al., 1983, Gene 26: 25-39. Theglands are ground to a fine powder in liquid nitrogen using a pestle andmortar. The powder is added to five volumes of a 1:1 mixture of watersaturated phenol and NETS [20 mM Tris pH 7.8, 200 mM NaCl, 2 mM EDTA, 1%SDS] at 85° C. and mixed until homogenous. The mixture is cooled,centrifuged and the upper aqueous phase is recovered. The phenol layeris re-extracted with a half volume of NETS at 85° C. and the pooledaqueous phases is re-extracted with aqueous phenol. Nucleic acids areprecipitated out of solution by the addition of two volumes of ethanoland are collected by centrifugation. This preparation is enriched forsingle stranded nucleic acids by two rounds of LiCl precipitation. Threevolumes of 4M LiCl are added to the dissolved RNA preparation and themixture is incubated on ice for 1 hour. The precipitate is recovered,washed with 70% ethanol, dried and the whole procedure is repeated.Poly-A RNA is selected from this enriched RNA fraction by two rounds ofpurification on an oligo-dT cellulose column [Collaborative ResearchInc., type 3] as described by the manufacturer. Messenger RNA isconcentrated by ethanol precipitation and is re-dissolved in steriledistilled water.

Approximately 10 ug of poly-A RNA are converted into double strandedcDNA using the Bethesda Research Laboratories Inc. cDNA synthesis kitaccording to the manufacturer's instructions. The cDNA is methylated toprotect internal EcoRI restriction sites. The synthetic linkerCGGAATTCCG [New England Biolabs #1004] containing the EcoRI restrictionsite is phosphorylated with polynucleotide kinase and it is ligated tothe cDNA with T4 DNA ligase. The ligated DNA is digested with EcoRI tocreate cohesive ends and the cDNA is size fractionated on a SephacrylS-500 column [Pharmacia]. The cDNA was ligated to dephosphorylatedlambda gt11 arms [Promega Biotec] and packed into phage particles invitro using the Packagene system [Promega Biotec] according to themanufacturer's instructions. These phage particles constitute the cDNAlibrary.

An oligonucleotide [ACGATGCAAATAACTCAG] corresponding to the N-terminalamino acid sequence of the antigen (see FIG. 1) is synthesized and usedto screen the cDNA library for full length clones. The lambda gt11 cDNAlibrary is screened by the plaque hybridization method of Benton andDavis, Science, 196, 180-182 (1977), and Maniatis, using theoligonucleotide probe labelled with ³² P.

The library is plated on 160 mm NZCYM agar plates in NZCYM top agar at10⁴ p.f.u./plate and incubated at 37° C. for 10-15 hours. Filterreplicas of the plates are taken with nitrocellulose filters (BA85,Schleicher and Schuell) and processed according to Maniatis.

Positive lambda gt11 phage clones are picked and replated and rescreenedto ensure homogeneity. Purified phage are prepared from several positiveclones, and the recombinant phage DNA is isolated and the DNA insertsubcloned into a plasmid vector, Bluescript SK (Stratagene).

EXAMPLE 2 Cloning of the 67kDa glycoprotein gene from Theileriapiroplasm genomic DNA

T. parva parva (Muguga) piroplasms were isolated as previously describedin Mack, S. R., 1978, J. Parasit. 64:166-168. For purification of DNA,piroplasms were suspended in 10 ml TNE (10 mM Tris-HCl pH 7.8, 100 mMNaCl, 1 mM EDTA). Sodium dodecylsulphate (SDS) and RNase A (boiled in 10mM Tris-HCl, 0.1 mM EDTA, pH 8 for 10 min at 93° C.) were added to finalconcentrations of 0.5% and 100 ug per ml, respectively. The suspensionwas incubated for 1 hour at 37° C. Proteinase K (BRL, Gettysburg, Md.,U.S.A.) was then added to a concentration of 100 ug per ml and thepreparation incubated for an additional 2-3 hours at 50° C. The lysatewas extracted once with phenol, twice with phenol:chloroform (1:1), andtwice with chloroform:isoamyl alcohol (24:1) before precipitation of DNAwith two volumes ice cold 100% ethanol. DNA was pelletted at 500×g for15 min at 4° C., dried at 37° C. and dissolved in sterile TE buffer (10mM Tris, pH 8.0, 0.1 mM EDTA).

A library of T. parva parva (Muguga) genomic DNA fragments wasconstructed in the bacteriophage vector lambda gt11 (Promega Biotech) bymethods previously described. Young, R. A., et al., 1985, Nature,316:450-45. Approximately 2 ug of piroplasm DNA were sheared by passagethrough a 19 gauge needle, 250-300 times, to produce fragments of 3 to 8kb in size. The DNA was methylated with EcoR I methylase using reactionconditions described by the manufacturer (New England Biolabs, Beverly,Mass., U.S.A.). Treatment with Klenow fragment of DNA polymerase andligation with EcoR I linkers was carried out as described in Maniatis.The excess DNA was digested twice, for 2 h at 37° C., with 100 unitsEcoRI, followed, each time, by phenol-chloroform (1:1) extraction andexcess linkers removed by passage through a Sephacryl S-300 spin column(Pharmacia, Uppsala, Sweden). The DNA fragments were then mixed with 0.5ug of dephosphorylated lambda gt11 arms at a 1:2 molar ratio of insertsto arms and ligated using T4 DNA ligase. The recombinant DNA was thenpackaged into phage particles using commercially available extracts(Promega Biotech) and the resultant phage amplified by plating on E.coli strain YlO9O. Approximately 1.1×10⁶ recombinant phage were producedfrom 0.1 ug insert DNA in a library that contained 85% recombinants.

An oligonucleotide [ACGATGCAAATAACTCAG] corresponding to the N-terminalamino acid sequence of the antigen (FIG. 1) was synthesized and used toscreen the genomic DNA library for full length clones. The lambda gt11genomic DNA library was screened by the plaque hybridization method ofBenton and Davis, 1977, Science, 196, 180-182, and Maniatis, using theoligonucleotide probe labelled with ³² P.

The library was plated on 160 mm NZCYM agar plates in NZCYM top agar at10⁴ p.f.u./plate (10,000 plaque forming units per plate) and incubatedat 37° C. for 10-15 hours. Filter replicas of the plates were taken withnitrocellulose filters (BA85, Schleicher and Schuell) and processedaccording to Maniatis.

Positive lambda gt11 phage clones were picked and replated andrescreened to ensure homogeneity. Purified phage were prepared fromseveral positive clones, and the recombinant phage DNA isolated and theDNA insert subcloned into plasmid vector pUC18 (Pharmacia).

EXAMPLE 3 Production of the 67 kDa Theileria Antigen in Bacterial Cells

The 67 kDA antigen is preferably expressed by manipulating full-lengthcDNA into expression vectors. However, it is possible to assemble fulllength expressible sequences from genomic DNA and partial cDNAsequences. Both methods are described below.

Two strategies may be used to express the Theileria antigen. The firstexpresses the complete gene sequence including the presumptive signalsequence that would not be present in the "mature" sporozoite antigen.The second method expresses sequences encoded by the "mature" geneproduct.

The lambda gt 11 full length cDNA clone, (Example 1) contains two EcoRIfragments, approximately 1220 and 990 bp long (FIG. 3). These areshotgun cloned into the EcoRI site of plasmid Bluescript SK(Stratagene). The Bluescript recombinants (plasmid 1 and 2, see FIG. 3,which contain the 990 bp and 1220 bp DNA fragments, respectively), areused as the source of cDNA in constructing the expression recombinants(FIGS. 3 and 5).

A. Expression of the complete gene product

The gene contains a BclI site 23 nucleotides in from the N-terminal end(FIG. 1). Since BclI digestion is blocked by the dam methylase, plasmid2 is grown in a methylase-deficient strain of E.coli such as NK5772.Plasmid 2 is prepared from NK5772 and is digested with BclI and asynthetic adaptor is attached to the DNA.

    5' CGGATCCCGATGCAAATAACTCAGTTTTTGCT 3'

    3' GCCTAGGGCTACGTTTATTGAGTCAAAAACGACTAG 5'

The adaptor contains a BamHI site at the 5' end. The ligated DNA isdigested with BamHI and EcoRI, the 1200 bp DNA is purified and thencloned into pGEX3 (Smith, D. B. & Johnson, K. S., 1988, Gene 67:31-40;Medos Company Pty. Ltd.) to give plasmid 3. The remainder of the genewhich is encoded on the 990 EcoRI fragment of plasmid 1 is cloned intothe EcoRI site of plasmid 3 and recombinants containing the 990 bpfragment in the correct orientation are isolated. The resultingexpression plasmid, plasmid 4, is transformed into an E.coli strain,such as JM109.

The Theileria antigen is purified as described below.

B. Expression of the "mature" gene product

To express the "mature" Theileria 67 kDa antigen, i.e. lacking thesignal sequence, step 2 in FIG. 3 is varied as follows. Plasmid 2 isdigested with BclI and subjected to limited Bal 31 digestion. BamHIlinkers [New England Biolabs #1017] are attached. The DNA is digestedwith BamHI and EcoRI and the 1200 bp DNA is purified. This DNA is clonedinto pGEX3 and the recombinants are sequenced to determine the extent ofthe Bal 31 deletions. Clones containing deletions ending at nucleotidenumber 54, 57 and 60 (FIG. 1) are kept and processed.

Plasmid 1 is digested with EcoRI, the 990 bp DNA is purified and clonedinto plasmid 3. Recombinants are screened to isolate clones containingthe 990 bp EcoRI fragment in the correct orientation, plasmid 4. Thisplasmid is transformed into an E. coli strain, such as JM109.

To achieve synthesis of the Theileria antigens in E. coli, cultures ofJMI109 carrying the expression plasmid are grown in rich medium (e.g., Lbroth), containing ampicillin to maintain the plasmid, at 30°-37° C. tointermediate cell density. IPTG is then added to induce expression ofthe recombinant gene from the tac promoter.

The fusion protein is affinity purified from E.coli lysates as describedby the manufacturer (Medos Company Pty. Ltd.). This exploits theproperties of Sj26, which is encoded by pGEX3 and to which the Theileriaantigen is fused. Sj26 is a glutathione-S-transferase which has a highaffinity for glutathione. The fusion protein is purified usingglutathione-agarose beads and eluted with free glutathione. PureTheileria antigen is recovered from the fusion protein by cleavage withFactor Xa which cleaves at the fusion site. By passing the cleavageproducts through the affinity column, Sj26 is retained on the column andpure Theileria antigen is isolated.

C. Assembly of a prokaryotic expression plasmid from partial theileriacDNA sequences and genomic DNA

A complete 67 kDa antigen encoding segment was assembled from a genomicDNA clone and from a partial cDNA clone according to FIG. 4. The 2900 bpand 2400 bp EcoRI fragments from the genomic clone and the 800 bp EcoRIfragment from the cDNA clone were shotgun cloned into pUC18 (Pharmacia).The 67 kDa gene spans the two genomic DNA fragments. The recombinantplasmid carrying the 2900 bp insert contains an intron which is locatedbetween the PstI and EcoRI sites (see FIG. 4). The partial cDNA clonecontains sequences from this EcoRI site to beyond the PstI site. Thegenomic PstI-EcoRI fragment was replaced with the cDNA PstI-EcoRIfragment thereby removing the intron.

The above plasmid also has a BclI site 23 nucleotides in from theN-terminus of the 67 kDa gene (see FIG. 1). For reasons describedearlier, the recombinant plasmid was grown in E. coli strain NK 5772.Purified plasmid was digested with BclI and a synthetic adaptor wasligated to the ends. The adaptor contains a BamHI site at the 5' end andthe 22 nucleotides 5' of the BclI site.

    5' CGGATCCCGATGCAAATAACTCAGTTTTTGCT 3'

    3' GCCTAGGGCTACGTTTATTGAGTCAAAAACGACTAG 5'

The ligated DNA was digested with BamHI and PstI and the 500 bp DNA waspurified. The pUC18 recombinant containing the cDNA insert was digestedwith PstI and EcoRI and the 600 bp DNA purified. The recovered DNA wasligated to pGEX3 digested with BamHI and EcoRI. The remainder of the 67kDa gene which is on part of the 2400 bp genomic DNA was cloned into thepGEX3 recombinant to give a construct that expresses the complete 67 kDagene product.

This pGEX3 recombinant, however, contains excess genomic DNA. This extraDNA was deleted by the following procedure. Plasmid DNA was digestedwith StuI, which cleaves the insert DNA 169 bp downstream of the stopcodon of the 67 kDa gene. BamHI linkers were attached to the ends, theligated DNA was digested with BamHI and the larger 2300 bp DNA fragmentwas isolated. The recovered DNA was ligated back into pGEX3 digestedwith BamHI and recombinants containing the insert in the correctorientation were isolated (FIG. 4). This construct also expresses thecomplete 67 kDa antigen.

For convenience the deposited plasmid, phTpp(mug)-p67sp, provides this67 kDa antigen encoding segment in a kanamycin resistant plasmid pK19[Gene, 56:309-312, 1987 and available from CIBA GEIGY, Basle,Switzerland]. The segment is readily excisable using BamHI (FIG. 2). Theposition of two additional restriction enzyme sites is shown.

EXAMPLE 4 Production of the 67 kd Theileria Antigen in S. typhimurium

Two strategies are used to express the 67 kDa antigen in Salmonella. Thefirst involves transformation of Salmonella with an expression plasmidcontaining the gene. The second method involves introduction of the geneinto the chromosome of the Salmonella.

A. Transformation of Salmonella with an expression plasmid carrying thegene encoding the 67 kDa antigen

Most E.coli cloning vectors will replicate in Salmonella spp. Theinstability of some of the vectors can be countered to an extent bymaintaining a selection on the plasmid in Salmonella by inclusion ofantibiotic in the growth medium. The pGEX3 recombinant used to expressthe Theileria 67 kDa antigen in E.coli (see FIGS. 3 and 4) istransformed into avirulent (aroA) Salmonella typhimurium and expressionof the fusion protein is monitored by Western blotting.

B. Integration of the Gene encoding the 67 kDa antigen into Salmonella

To overcome the problem of plasmid instability in Salmonella, the geneencoding the 67 kDa antigen is inserted into the chromosome of theSalmonella host, using a system based on the his operon of Salmonella,(Hone, D. et al., 1988, A Chromosomal Integration System forStabilization of Heterologous Genes in Salmonella Based Vaccine Strains,Microbial Pathogenesis, Vol. 5, pp. 407-418. In this case, a hisOGdeletion mutation is first introduced into the S. typhimuriumchromosome, and then replaced by introducing a plasmid containing thecomplete hisOGD region plus the DNA encoding the 67 kDa antigen. Byhomologous recombination, the introduced (complete hisOGD region plusDNA encoding the 67 kDa antigen) DNA will replace the hisOG deletionmutation. Recombinants can be selected His⁺.

The plasmid pADE 172 carries the hisOGD region minus the his regulationsequence (hisO) and part of the hisG gene. This plasmid is transformedinto the S. typhimurium strain. Strains in which the deleted his regionhas replaced the chromosomal his sequences are isolated by replicaplating on nutrient agar and M9 agar. (Strains carrying a deleted hisregion are His⁻ and grow on the former but not the latter.) The hisstrain is then cured of resistant plasmids by standard methods, to allowtransformation with another plasmid.

The plasmid pADE 172 contains the complete his OGD sequences. The cDNAencoding the 67 kDa antigen is inserted upstream of his0, and therecombinant plasmid introduced into the his strain of S. typhimurium.Recombinant strains, which are His , are selected on M9 agar, cured ofthe resident plasmids and tested for expression of the 67 kDa antigen byWestern blotting. Confirmation of chromosomal integration is achieved bypreparation of chromosomal DNA from recombinant strains and analysis byDNA blotting with the 67 kDa gene.

EXAMPLE 5 Expression of 67 kDa Antigen in Mouse Cells

Expression of the 67 kDa antigen in mouse cells can be achieved usingeither the full length cDNA clone (FIG. 5) or the gene assembled fromgenomic and cDNA sequences (FIG. 6).

To express the full length cDNA sequence plasmid 1 and plasmid 4,constructed as described in FIG. 3, are digested with BamHI and KpnI andthe 600 and 1500 bp inserts which are released are recovered and ligatedto pMT010/A⁺. (Choo, K. H. et al. DNA 5:529-539, 1986; the plasmid waskindly provided by Dr. Choo) digested with BamHI alone. Recombinantplasmid containing both inserts in the correct orientation is isolatedand used to transfect mouse cells (FIG. 5).

To express the gene assembled from genomic DNA and cDNA sequences (seeFIG. 4) the pGEX3 recombinant or dependent plasmid, phTpp(mug)-p67sp(see FIG. 2), is digested with BamHI and the 2300 bp insert which isreleased is recovered and cloned into pMT010/A⁺. Recombinant plasmidcontaining the insert in the correct orientation is isolated and used totransfect mouse cells (FIG. 6).

Mouse LTK-cells expressing the Theileria antigen are isolated asdescribed by Choo, et al., DNA 5:529-59 (1986). The cells are grown inDulbecco's Modified Eagle's medium supplemented with 10% foetal bovineserum and the cells are transfected with recombinant plasmid using thecalcium phosphate precipitation method. The cells are cultured for 48hours before selection of G418 (GIBCO Laboratories) resistance.Surviving transformant cells are pooled and subjected to stepwiseselection in methotrexate, to co-amplify the cloned Theileria gene.Expression of the Theileria antigen from the metallothionein promoter isincreased by the addition of zinc to the growth medium.

EXAMPLE 6 Production of vaccinia viral particles containing geneencoding the 67 kDa antigen

The strategy involved in obtaining recombinant vaccinia viral particlesencoding the 67 kDa antigen comprises of two steps. The first is toinsert the DNA encoding the 67 kDa antigen into a suitable plasmid. Thesecond step involves transfection of the plasmid into mammalian cellswhich have been infected with vaccinia virus. Incorporation of the DNAencoding the 67 kDa antigen into the genome of the virus occurs byhomologous recombination. Positive recombinants are selected, grown inmammalian cell cultures and purified for inoculation into cattle.

a) Construction of plasmid

A plasmid suitable for use in this system is pGS62. Langford, C. J., etal., 1986, Mol. Cell. Biol. 6, 3191-99. The essential features of thisplasmid are: i) a multiple cloning site containing BamHI, SmaI and EcoRIsites for insertion of foreign genes, ii) the P7.5 promoter of vacciniato direct synthesis of the inserted gene and iii) segments of thevaccinia TK gene flanking both ends of the foreign gene to directhomologous recombination of the foreign gene plus TK flanking sequencesinto the TK gene of vaccinia virus. The BamHI DNA fragment encoding the67 kDa antigen, constructed either from full length cDNA (FIG. 5) or byassembling a hybrid gene from genomic and cDNA sequences (FIGS. 2 and 4)is inserted into pGS62 at the BamHI site and recombinants containing theinsert in the correct orientation are isolated.

b) Production of recombinant virus

The mammalian cells, such as 143 TK⁻ cells are grown as a monolayer toconfluency, and inoculated with 0.05 p.f.u. of virus per cell. Onemicrogram of the recombinant plasmid is added to 19 ug of carrier DNA in1.0 ml HEPES-buffered saline and precipitated by addition of CaCl₂ to afinal concentration of 125mM, at room temperature for 30 min. Two hoursafter addition of the virus, the virus inoculum is removed and themonolayer washed twice with medium. The DNA suspension is added to themonolayer and incubated at room temperature. After 30 min, 5ml of mediumcontaining 5% foetal bovine serum is added and the cells are incubatedat 37° C. for a further 3.5 hours.

The cells are washed and incubated in medium with 5% foetal bovine serumfor 48 hrs. The cells are collected and virus progeny are released bythree cycles of freeze thawing. To select for recombinant viruses, 143TK⁻ cells are inoculated with the virus progeny. One to two hours afteraddition of the virus, the medium containing the virus inoculum isremoved and replaced with medium containing 1% low gelling temperatureagarose, 5% foetal bovine serum and 25 ug/ml 5-bromodeoxyuridine. After48 hrs the monolayer is stained with neutral red to locate virusplaques. These plaques can be selected and amplified for use in a secondround of screening. Two cycles of plaquing usually produce a homogeneousviral stock. The presence of the gene encoding the 67 kDa antigen can beconfirmed using DNA blotting in which viral DNA is probed with plasmidcontaining the cDNA encoding the 67 kDa antigen. Expression of the 67kDa antigen in infected cells can be confirmed by immunoblotting usinglysates of virus infected cells and probing with monoclonal antibodiesspecific for the 67 kDa antigen.

EXAMPLE 7 Immunoreactivity of recombinantly produced 67 kDa antigen inE.coli

Groups of rats have been immunised with two pGEX fusion proteinsexpressing different regions of the Theileria 67 kDa antigen. Group Ireceived the control Sj26 protein. Group II was immunised with a fusionprotein encoding amino acid residues 9-316 (FIG. 1) of the Theileriaantigen and group III was immunised with a fusion protein encoding aminoacid residues 397-709 of the Theileria antigen.

Each rat was inoculated with 5 ug of protein in complete Freund'santigen as the primary dose. The rats were boosted twice at two weekintervals with 5 ug of protein in incomplete Freund's antigen andsacrificed two weeks after the third inoculation.

Sera taken from rats in groups II and III recognise the Theileria 67 kDaantigen on Western blots. Furthermore, the sera from these animalscompletely neutralise sporozoite infectivity in the in vitro assaysystem. Control sera from group I rats fail to recognise the Theileriaantigen and fail to neutralise sporozoite infectivity.

The above results show that the presence of carbohydrate sidechains onthe Theileria antigen are not essential for evoking neutralisingantibodies.

Since rats were immunised with two non-overlapping regions of theantigen, there is more than one epitope exposed on the surface of thesporozoite.

Rats have not been immunised with the complete recombinant product,although such a construct is available. The region between residues 316and 397 were not included in the above experiments.

Plasmid pGEX is a family of three vectors allowing expression of DNA inthe three different reading frames. The results described above usedpGEX1 and pGEX3.

EXAMPLE 8 Immunization protocol

Areas of Eastern Africa where the disease is prevalent can experience anabundance of ticks after receiving sufficient rainfall. Under thesecircumstances it is desirable that animals are vaccinated before therains.

The preferred age of vaccination of calves would be 2 months whilevaccination for older stock is done at any time. For both calves andadults the priming dose, composed of 1.0 mg of purified antigen producedby recombinant DNA technology and 3 mg of saponin in 1 ml of saline,would be administered subcutaneously in an area cranial to theprescapular lymph-node, followed by similar booster doses 4 to 8 weekslater. Revaccination should be done semi-annually, particularly foranimals under heavy challenge and preferably just before the rains.

What is claimed is:
 1. A substantially pure and isolated antigenic polypeptide having the amino acid sequence set forth in FIG. 1 and characterized by inducing immunoprotection against infection by Theileria parva in bovine animal species when administered to the animal in an amount effective to induce the immunoprotection.
 2. A vaccine for inducing immunoprotection in bovine animal species against infections of Theileria parva comprising pharmaceutically acceptable excipients and a substantially pure and isolated antigen having the amino acid sequence set forth in FIG. 1 said antigen present in an amount effective to induce immunoprotection against infection by Theileria parva when administered to the animal.
 3. A vaccine of claim 2 wherein the antigen is glycosylated.
 4. A vaccine of claim 2 wherein the antigen is not glycosylated.
 5. A vaccine of claim 2 wherein the animals are cattle.
 6. A vaccine of claim 2 wherein the antigen is a recombinant protein.
 7. A vaccine of claim 2 wherein the substantially pure and isolated antigen is at least 75% pure.
 8. A method for protecting bovine animal species from infection by Theileria parva comprising the administration of a vaccine comprising an amount of a substantially pure and isolated antigen having the amino acid sequence set forth in FIG. 1 said amount effective for inducing immunoprotection against infection by Theileria parva in the animal.
 9. A method of claim 8 wherein the antigen is glycosylated.
 10. A method of claim 8 wherein the antigen is not glycosylated.
 11. A method of claim 8 wherein the animals are cattle.
 12. The method of claim 8 wherein the administering step includes parenteral administration.
 13. A method of claim 8 wherein the antigen is a recombinant protein.
 14. A method of claim 8 wherein the substantially pure and isolated antigen is at least 75% pure. 